Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning.
Differential display (DD) has been used extensively to detect differentially expressed genes. However, the low reproducibility of displayed bands makes verification by Northern blot difficult and the technique is labor-intensive. This report describes a fluorescent DD with a ROX (carboxy-X-rhodamine)-labeled anchor primer and a revised RT-PCR, utilizing AMV reverse transcriptase, a more thermostable reverse transcriptase than Mu-MLV, and optimized concentrations of dNTPs and of MgCl2. Our technique yielded clear fingerprints with high reproducibility. Further, we have developed a method of rapid screening to select the cDNA fragments of interest in excised bands from a polyacrylamide gel without cloning. This method consists of electrophoresis with an agarose gel containing a base-specific DNA ligand to separate the equally sized fragments differing in base composition, and side-by-side comparison of the reamplified products from the experimental and control lane. Most of the cDNA fragments selected by this protocol provided readable sequences by direct sequencing and were confirmed to exhibit differential expression by Northern blot analysis or semiquantitative RT-PCR.
Yoshikawa
Y
Y
Advanced Biomedical Center, Takara Shuzo Co., Ltd., Shiga, Japan. tabc-uno@mx.biwa.or.jp
Mukai
H
H
Asada
K
K
Hino
F
F
Kato
I
I
D001483:Base Sequence; D016172:DNA Fingerprinting; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D012235:Rhodamines
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012235:Rhodamines